In order to understand bacterial pathogenesis, it is essential to have a good understanding of bacterial physiology. One area that is particularly lacking in information is the role of calcium in prokaryotes. The long-term goal of this research project is to gain an understanding of the function of calcium in bacteria. Of particular interest are regulatory functions of calcium and potential connections between calcium and bacterial pathogenesis. The marine bacterium and significant human pathogen Vibrio parahaemolyticus is an ideal model system for this study. This organism occupies environments that differ substantially in calcium content, for example, seawater compared to the human gastrointestinal tract. Significantly, preliminary results suggest that calcium affects a number of V. parahaemolyticus cellular processes, including processes that may play a role in pathogenesis. In addition, results strongly suggest that calcium regulates gene expression in this organism. Three specific aims are outlined in this proposal. Aim 1 is designed to identify gene products involved in calcium sensing and response. An existing strain containing a calcium-regulated lux fusion will be mutagenized using a transposon to identify insertions that disrupt calcium regulation. Experiments outlined in Aim 2 are designed to determine the extent of calcium-regulated gene expression in V. parahaemolyticus and identify regulated genes. Two-dimensional gel electrophoresis will be used to analyze protein expression in response to altered calcium levels. Mutagenesis with a transposon containing a promoterless reporter gene will allow identification of genes that are calcium-regulated. The goal of Specific Aim 3 is to thoroughly investigate the phenotypic effects of calcium in wild type V. parahaemolyticus and to phenotypically characterize the mutants isolated in Aims 1 and 2. Together, these experiments are expected to provide significant insight into the role of calcium in bacteria, thereby filling a gap in the current understanding of bacterial physiology. This study may increase the understanding of the role of calcium in bacterial pathogenesis. Finally, as this proposal is based primarily on a genetic approach, this project will provide ideal undergraduate research projects for students at Luther College.